Our laboratory is interested in the molecular mechanisms that link protein absorption and gene regulation in proximal tubule and which are likely to participate in the pathology seen in many proteinuric renal diseases. We have identified two signaling pathways that appear to provide this link. One pathway uses regulated proteolysis of the receptor megalin while the other pathway depends on the activity of a protease called ADAM10. The megalin pathway appears to inhibit brush border gene products while the ADAM10 pathway activates similar genes. The objective of this research project will be to characterize in detail the molecular mechanisms that contribute to the ADAM10 signaling pathway and to identify the genes regulated by it. Toward these ends we will first characterize ADAM10 protein in the rodent proximal tubule. This will be accomplished using specific antibodies in conjunction with state-of-the-art, immunocytochemical and biochemical methods. Secondly, we will identify the ADAM10 substrate that is cleaved in the proximal tubule. This will be accomplished both by screening known substrates from other cell types in kidney as well as by performing proteolytic studies of renal proteins using recombinant ADAM10. Finally, we will directly study ADAM10 activity in proximal tubule in a transgenic mouse model. In these studies we will target and over-express in the proximal tubule either active ADAM10 or an inactive mutant. Targeting to the proximal tubule will be accomplished using the promotor for the Sglt2 gene. These animals will be developed at Yale using the in-house facilities. The phenotype of the transgenic mice, as it relates to renal function, will be evaluated using the facilities available in the Nephrology Section at Yale Medical School. Chronic kidney disease, resulting in large part to epidemic levels of diabetes in the United States, is a growing health problem. Elevated urinary protein is one of the most significant predictors of renal disease and controlling proteinuria in the patient with proteinuric kidney disease is paramount for the physician. The study proposed here will provide important insights into the effect of urinary protein on renal function that may lead to improved strategies for treating patients with chronic kidney diseases.